Abstract: Introduction. Collagen plays a major role in tissue repair and is a valid option for the treatment of chronic and acute wounds. Collagen speeds fibroblast deposition inside the extracellular matrix and stimulates angiogenesis, granulation tissue formation, and remodeling. Evaluation of the efficacy of wound treatment can be made by noninvasive, objective instrumental assessment. Materials and Methods. Forty-six patients with venous leg ulcers were enrolled into the study. The ulcers showed no clinical signs of healing over the course of 6 weeks despite standard treatment. Patients in group A were treated with a biological dressing made of heterologous collagen (Condress®, Abiogen Pharma, Italy) and with a standard treatment in group B. The duration of the treatment was 4 weeks. Chronic wounds were monitored by means of noninvasive assessment using a laser scanning system capable of performing 3D evaluation and color defragmentation. Results. The median increase in granulation tissue after 4 weeks of therapy was 65% in group A compared to 7% in group B (P < 0.001). A median reduction of 50% in relative ulcer area was observed in group A after 4 weeks of treatment, compared with 32% in group B (P < 0.05). Conclusion. This study objectively demonstrated that a heterologous collagen biological dressing induced granulation tissue in hard-to-heal venous leg ulcers better than standard treatment. The scanning system used to monitor lesions was not only fast, but was easy to use, and had good intra- and inter-observer reproducibility. Collagen plays a relevant role in cutaneous tissue repair and represents a valid therapeutic option when used as a bioactive advanced dressing in chronic wound management. It improves fibroblast deposition in the dermal matrix and stimulates angiogenesis, granulation tissue formation, and reepithelization.1 Fibroblasts mainly participate in the biosynthesis of collagen, which acts as a mold, precursor, plastic material, and cementing substance in the wound healing process. The treatment efficacy of collagen products requires precise monitoring on skin lesions using objective, accurate, and reproducible methods. More recently a new laser scanning system was developed that provides 3D wound assessment and acquires colorimetric information from cutaneous lesions.2 The following is a report on the experience in using a heterologous equine collagen in hard-to-heal venous leg ulcers.

Materials and Methods

Forty-six patients (31 women, 15 men) attending the outpatient leg ulcer clinic at the Department of Dermatology, University Hospital (Santa Chiara, Pisa, Italy) with hard-to-heal venous leg ulcers of more than 6 weeks’ duration were enrolled into the study. Patients entering the study showed clinical and instrumental signs of venous insufficiency complicated by lower leg ulceration (mean size 36.4 cm2). Exclusion criteria were: leg ulcers of purely arterial etiology, ulcers with clinical signs of infection, known sensitivity to the tested product, and the unwillingness of a patient to attend the clinic regularly for treatment and assessment. Before the study began, patients were required to complete a 3-week run-in period with standard treatment, including 4 layers of compression bandaging and moist wound healing. At the end of the run-in period, patients with a wound size reduction > 50% were excluded from the study. Informed consent was obtained from all patients and ethical approval was received from the local ethical committee before initiating the study. Patients were alternately allocated by 1 of the investigators into 2 groups: • Group A was treated with a sponge of heterologous collagen dressing (Condress®, Abiogen Pharma, Italy) and short stretch compression. •Group B was treated with an interface inert dressing and short stretch compression. The compression system used in this study was made of 2 layers: a protective and absorbent natural cotton padding layer and a short stretch bandage layer. Dressings were changed twice a week in both groups for 4 weeks. Wound assessment. Wound size and percentage of granulation tissue were monitored weekly using a new integrated instrument called DERMA® system (CGS, Pisa, Italy [Figure 1]). The system allows the user to acquire accurate 3D digital models of wounds of various etiologies, and at the same time, gather information on wound surface and color. The instrument is provided with a laser triangulation scanner (V I 900, Minolta, Japan), which acquires the 3D wound geometry and captures red-green-blue (RGB) images aligned to the geometry, as well as gathering a color-based characterization of the lesion. The system provides a single and uniform interface to manage patient data, supports 3D scanning of the lesion region, and performs various geometric (on the 3D model) and colorimetric (on the RGB image) measurements and relative comparisons. All acquired data (3D geometries and images), as well as the measurements taken, are stored in a database for monitoring the evolution of the lesion over time. Color assessment. Color characterization is undoubtedly important in wound healing because the different colors that appear in a wound bed can give objective information on the status of the wound itself, according to the following classification: red, yellow, and black. When performing a clinical wound assessment, it is assumed that red is the color of granulation and indicates a wound that is healing well. Yellow is the color of fibrin—while it is a physiological tissue repair constituent, if it is present in large quantities, it can slow and possibly impede healing. Black is the color of necrosis and indicates that the wound is in an involutional phase. The DERMA system is able to calculate the percentage of each area with a corresponding color within the wound, thanks to region-growing algorithms, which starting from the image identify corresponding areas with similar colors and certain basic tones that the user chooses. The wound bed color evaluation starts when the user selects a point (seed) inside the previously defined wound edge and matches it to the color system scale in order to automatically differentiate the wound bed color features. Automatically a region-growing algorithm with absolute color classification criteria delimits the corresponding area. The user may use an automatic extension function that searches for other seeds that are compatible with the average color of the first region by iteratively applying the region-growing algorithm, or he/she may manually select another seed in an area that is thought to have similar color features (the region-growing algorithm is applied in this case as well). The user may control the “tolerance” of the region-growing algorithm step by step. The final step includes the overall wound. The user may repeat the procedure described above for 5 classifications maximum: the next application of the region-growing algorithm avoids the possible overlapping of areas associated with different colors by positioning its edge on the point that is chromatically equidistant between 2 “competing” seeds; the user may freely assign a name to each classification obtained, then the environment automatically calculates perimeter, area, and percentage related to the wound surface of each classification. The classification results are visualized with “false colors” corresponding to the average color of the classified region on the 3D wound model. The area outside the edge of the region simultaneously assumes a neutral color. Statistical analysis. Statistical data analyses were made using software for statistical evaluations (SPSS for Windows, version 11.0.0). The improvement differences found between the study groups were tested using analyses of variance (ANOVA). Additionally, the paired sample t-test was applied to determine whether improvement differences among wound evaluation parameters existed within each group. P < 0.05 was considered significant in both ANOVA and t-test.

Results

Forty-three subjects (93%) completed the study; 3 subjects (6%) withdrew from the study (1 in group A was lost to follow-up and 2 in group B requested withdrawal). In the comparison of final data at the conclusion of 4 weeks, a significant difference was observed in the color parameter between group A and group B (Figures 2–4). The median increase of wound bed granulation tissue after 4 weeks of therapy was 65% in group A compared to 7% in group B (P < 0.001). A median reduction of 50% in relative ulcer area was observed in group A at the conclusion of treatment compared with 32% in group B (P < 0.05). In this study, the DERMA system allowed for standardized 3D image acquisition, color analysis, and automatic measurement of the lesion surface. System precision was assessed by comparing measurements of clearly known objects and the results were found to be positive: linear error was < 0.2 mm; surface error was < 3%; and volume error was close to 5%.

Discussion

The primary aim of the study was to evaluate wound healing objectively after the application of a heterologous collagen sponge dressing and acquire a quantitative and qualitative comparison through observing objective data. Collagen is the main structural protein component of connective tissue and represents about a quarter of the entire proteic patrimony of the organism.3 All connective tissues are composed of 2 major constituents: cells and extracellular material. Collagen is a vital structure in wound healing and is essential when cross-linked for wound tensile strength. The role of collagen involves stimulating fibroblast activity4 and improving the healing process. The most important functions of fibroblasts are synthesis and deposition of the extracellular matrix components.5 Fibrous connective tissue elements include collagen, elastin, and reticulin, while the nonfibrous portion includes basic substances that are primarily water, salts, and glycosaminoglycans. Collagenase and other proteolytic enzymes are produced during the inflammatory phase and throughout the proliferative phase as fibroplasia regulators.6 The goals of this phase of healing involve filling in the wound defect with new tissue and restoring skin integrity.7 Dressings composed of a collagen matrix for clinical applications with major considerations of biosecurity are available. The application of equine collagen to the wound, besides having an important hemostatic activity, promotes fibroblast and keratinocyte migration and leads to more effective tissue healing.8 As a result of the repair properties of collagen, much research has been done to verify a possible acceleration of the tissue repair process by applying collagen to lesions.9 In this study, the collagen sponge proved to be efficacious in the promotion and stabilization of granulation tissue. Noninvasive measurement techniques are useful in therapeutic protocol validation with advanced biologic dressings.10 The collagen available for this use comes in the form of soft sponges, which are lyophilized and sterilized. It has been proven that the material implanted, lysed by enzymatic digestion by leukocytic proteases, maintains intimate contact with the bottom of the lesion, is embedded into the granulation process, and forms a plastic scaffolding over which fibroblast migration takes place followed by endogenous cell invasion. The result is a physiological and natural tissue repair. Its spongy network contributes to exudate absorption and blocking of possible extensions of the wound, preventing bacterial growth that would delay the healing process. Moreover, the pores are neither so large as to favor dryness nor too small that they hinder granulation or gaseous exchange. It would appear that aside from acting as a mechanical support and agent of fibroblast motility, heterologous collagen participates as a feeding substrate to the metabolic activity of the granulation tissue. The product acts locally without absorption and does not enter into the systemic structures of the body. Rather, it enters the fibroblastic cellular local metabolism, where it acts by stimulating the production of endogenous collagen, which is responsible for wound healing. The wound healing process involves phenomena such as proliferation, migration, and cell differentiation—all of which are influenced by the presence of collagen. In the final stage of repair with a continuity tissue solution, the maturation of collagen takes place and collagen fibers create a bridge between the edges of the damaged tissues, going on to form or favor a scar with elasticity and mechanical strength.

Conclusion

The possibility of positively influencing the repair of damaged tissue has long been studied in dermatology, plastics, and surgery. Objective and reproducible measurement of repair process phases is becoming increasingly interesting and may soon represent a valid tool for physicians and medical staff. New diagnostic and therapeutic tools will become helpful in the fields of research and clinical practice, offering an increasing amount of information on tissue repair processes. These tools will provide innovative methods for collecting, storing, and comparing data over time. One must hope for close collaboration between clinical experts and biotechnological engineers so that together they might develop new, noninvasive technologies that will contribute to the progression of clinical practice and research.